Insulin is a proven therapy for the treatment of juvenile-onset diabetes and later stage adult-onset diabetes. The peptide is biosynthesized as a larger linear precursor of low potency (approximately 2% to 9% of native insulin), named proinsulin. Proinsulin is proteolytically converted to insulin by the selective removal of a 35-residue connecting peptide (C peptide). The resultant heteroduplex formed by disulfide links between the insulin “A chain” (SEQ ID NO: 1) and “B chain” (SEQ ID NO: 2) chain, representing a total of 51 amino acids, has high potency for the insulin receptor (nM range). Native insulin has approximately one hundredfold selective affinity for the insulin receptor relative to the related insulin-like growth factor 1 receptor, but demonstrates little selectively for the two different insulin receptor isoforms, named A & B.
Incretins are a group of gastrointestinal hormones that that are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the regulation of food intake. Pre-proglucagon is a 158 amino acid precursor polypeptide that is processed in different tissues to form a number of different peptides. Incretins include a number of proglucagon-derived peptides, including glucagon (SEQ ID NO: 701), glucagon-like peptide-1 (GLP-1; amino acids 7-37 are provided as SEQ ID NO: 703 and amino acids 7-36 as SEQ ID NO: 704), glucagon-like peptide-2 (GLP-2; SEQ ID NO: 708) and oxyntomodulin (OXM; SEQ ID NO: 706).
Glucagon is a 29-amino acid peptide that corresponds to amino acids 33 through 61 of pre-proglucagon, while GLP-1 is produced as a 37-amino acid peptide that corresponds to amino acids 72 through 108 of pre-proglucagon. GLP-1(7-36) amide (SEQ ID NO: 704; the C terminus is an arginine amide) or GLP-1(7-37) acid (SEQ ID NO: 703; C terminus is a glycine) are biologically potent forms of GLP-1, that demonstrate essentially equivalent activity at the GLP-1 receptor.
Glucagon is a life-saving medicine that is used in the acute treatment of severe hypoglycemia. Oxyntomodulin has been reported to have pharmacological ability to suppress appetite and lower body weight. Clinical studies with GLP-1 receptor agonists or stabilized GLP-1 analogs have proven this family of peptides to be an effective treatment for Type II diabetes.
In addition, gastric inhibitory polypeptide (GIP) is also known as a glucose-dependent insulinotropic peptide, and is a member of the secretin family of hormones. GIP is derived from a 153-amino acid proprotein encoded by the GIP gene and circulates as a biologically active 42-amino acid peptide (SEQ ID NO: 707). The GIP gene is expressed in the small intestine as well as the salivary glands and is a weak inhibitor of gastric acid secretion. In addition to its inhibitory effects in the stomach, in the presence of glucose, GIP enhances insulin release by pancreatic beta islet cells when administered in physiological doses. GIP is believed to function as an enteric factor that stimulates the release of pancreatic insulin and that may play a physiological role in maintaining glucose homeostasis.
As disclosed herein conjugates are formed between an insulin peptide and an incretin, including for example a glucagon, GLP-1, or GIP agonist, a GLP-1/GIP co-agonist, a GLP-1/glucagon co-agonist or a glucagon/GLP-1/GIP triagonist, wherein the conjugate has agonist activity at both the insulin receptor and the corresponding incretin receptor. More particularly, the conjugation of a glucagon related peptide (e.g., GIP, GLP-1 or glucagon) is anticipated to produce a beneficial modification of the insulin peptide activity. For example, linking a peptide having agonist activity at the glucagon receptor to an insulin peptide is anticipated to enhance targeting of the conjugate to the liver since the glucagon receptor is predominately located in the liver. Targeting of the conjugate to the liver is desirable since the liver is primarily involved in glucose production not utilization. Thus targeting the liver may be a safer approach to shutting off glucose production than occurs when insulin contacts other tissues such as muscle or fat, where in addition to turning off glucose production it also stimulates glucose use leading to a higher risk of hypoglycemia. Also, there are glucagon receptors present on the alpha cells of the pancreas. Delivering the complex to the alpha cells may suppress additional glucagon production or make the alpha cell more sensitive to hypoglycemia. Applicants also anticipate that the presence of glucagon in the glucagon-insulin conjugates may serve as a buffer on the activity of the coupled insulin to provide a more baseline activity and thus avoid spikes in blood glucose levels.
Similarly, it is anticipated that conjugates of insulin peptides with other glucagon related peptides including the incretins GLP-1 and GIP and other related peptides having activity at the GLP-1 and/or GIP receptors will produce conjugates having beneficial properties. For example, GLP-insulin conjugate may be targeted to the hypothalamus, to decrease appetite as well as reduce blood glucose. Alternatively or additionally, the GLP-insulin conjugate may be targeted to the beta cells to drive anabolic response (increase islet beta cells' production of insulin).
The incretin-insulin peptide conjugates are also suitable for further structural enhancements that are envisioned to yield improved therapeutic index, through the use of prodrug chemistry; extended duration of action, by linkage of plasma proteins such as albumin, or other modifications, including pegylation and lipidation (e.g., addition of C14-C30 alkyl or acyl groups); and enhanced physical stability, by glycosylation. The preparation of single chain insulin analogs using a C-peptide linker also provides a novel structural location for where many of these chemical modifications can be successfully deployed. One use of the insulin conjugates disclosed herein would be in the treatment of diabetes while stimulating weight loss or preventing weight gain.